Compositions, systems, and methods for base diversification

Abstract

Described herein are methods of modifying or editing a target nucleic acid such as methods that edit cytosine to thymine and adenine to guanine and/or methods that edit cytosine to thymine, adenine, or guanine. Compositions and systems for modifying or editing a target nucleic acid are also described. Methods, compositions and systems described herein may be used for generating allelic diversity.

1 . A method of modifying a target nucleic acid, the method comprising:
 contacting the target nucleic acid with:
 a CRISPR-Cas effector protein 
 a guide nucleic acid comprising a RNA recruiting motif, 
 a cytosine deaminase, and 
 an adenine deaminase, 
   wherein the cytosine deaminase and the adenine deaminase concurrently and/or simultaneously modify the target nucleic acid optionally in a single delivery of reagents comprising the CRISPR-Cas effector protein, the cytosine deaminase, and the adenine deaminase, and/or   wherein the CRISPR-Cas effector protein and the cytosine deaminase and/or the adenine deaminase form a complex or are comprised in a complex, thereby modifying the target nucleic acid.   
     
     
         2 .- 6 . (canceled) 
     
     
         7 . The method of  claim 1 , wherein the cytosine deaminase and/or adenine deaminase comprise a MS2 capping protein (MCP) or a portion thereof. 
     
     
         8 .- 11 . (canceled) 
     
     
         12 . The method of  claim 1 , wherein the CRISPR-Cas effector protein comprises a peptide tag. 
     
     
         13 . The method of  claim 12 , wherein the adenine deaminase and/or cytosine deaminase comprise an affinity polypeptide capable of binding the peptide tag. 
     
     
         14 . The method of  claim 13 , wherein the cytosine deaminase and/or adenine deaminase is/are recruited to the target nucleic acid using the affinity polypeptide. 
     
     
         15 . The method of  claim 12 , wherein the peptide tag is recruited to the RNA recruiting motif via fusion to the MCP or portion thereof and the cytosine deaminase and/or adenine deaminase is/are recruited to the peptide tag using the affinity polypeptide. 
     
     
         16 . The method of  claim 1 , wherein the adenine deaminase and/or cytosine deaminase comprise a peptide tag. 
     
     
         17 . The method of  claim 16 , wherein the CRISPR-Cas effector protein comprises an affinity polypeptide capable of binding the peptide tag. 
     
     
         18 . The method of  claim 17 , wherein the CRISPR-Cas effector protein is recruited to the target nucleic acid using the affinity polypeptide. 
     
     
         19 . The method of  claim 16 , wherein the peptide tag is recruited to the RNA recruiting motif and the CRISPR-Cas effector protein is recruited to the peptide tag using the affinity polypeptide. 
     
     
         20 . The method of  claim 1 , wherein the CRISPR-Cas effector protein is a Type V CRISPR-Cas effector protein. 
     
     
         21 .- 41 . (canceled) 
     
     
         42 . A method of modifying a target nucleic acid, the method comprising:
 contacting the target nucleic acid with:
 a CRISPR-Cas effector protein, 
 a guide nucleic acid, and 
 a cytosine deaminase, 
   wherein the method modifies a cytosine (C) of the target nucleic acid to an adenine (A), guanine (G), or thymine (T), thereby modifying the target nucleic acid.   
     
     
         43 . (canceled) 
     
     
         44 . The method of  claim 42 , wherein contacting the target nucleic acid further comprises contacting the target nucleic acid with the CRISPR-Cas effector protein, guide nucleic acid, cytosine deaminase, and a uracil N-glycosylase (UNG). 
     
     
         45 . The method of  claim 42 , wherein the method comprises modulating DNA-binding affinity of the CRISPR-Cas effector protein. 
     
     
         46 .- 47 . (canceled) 
     
     
         48 . The method of  claim 42 , wherein the method comprises modulating residence time of the CRISPR-Cas effector protein at the target nucleic acid. 
     
     
         49 . The method of  claim 42 , wherein the method comprises generating an abasic site that is used as a template for a DNA polymerase. 
     
     
         50 . (canceled) 
     
     
         51 . The method of  claim 42 , wherein the method comprises inhibiting APE1. 
     
     
         52 . (canceled) 
     
     
         53 . The method of  claim 42 , wherein contacting the target nucleic acid further comprises contacting the target nucleic acid with the CRISPR-Cas effector protein, guide nucleic acid, cytosine deaminase, and a DNA ligase IV inhibitor. 
     
     
         54 . The method of  claim 42 , wherein contacting the target nucleic acid further comprises contacting the target nucleic acid with the CRISPR-Cas effector protein, guide nucleic acid, cytosine deaminase, and a DNA-PKcs inhibitor. 
     
     
         55 .- 56 . (canceled) 
     
     
         57 . The method of  claim 42 , wherein further comprising an exogenous polymerase that is fused to a Type V CRISPR-Cas effector protein. 
     
     
         58 . (canceled) 
     
     
         59 . The method of  claim 42 , wherein the CRISPR-Cas effector protein comprises a peptide tag. 
     
     
         60 - 93 . (canceled) 
     
     
         94 . The method of  claim 42 , wherein the target nucleic acid is present in a plant cell or eukaryotic cell.